title: Lyophilized insulin nanoparticles prepared from quaternized N-aryl derivatives of chitosan as a new strategy for oral delivery of insulin: in vitro, ex vivo and in vivo characterizations.
abstract: OBJECTIVE
  The purpose of this research was the development, in vitro, ex vivo and in vivo characterization of lyophilized insulin nanoparticles prepared from quaternized N-aryl derivatives of chitosan.
  
  
  METHODS
  Insulin nanoparticles were prepared from methylated N-(4-N,N-dimethylaminobenzyl), methylated N-(4 pyridinyl) and methylated N-(benzyl). Insulin nanoparticles containing non-modified chitosan and also trimethyl chiotsan (TMC) were also prepared as control. The effects of the freeze-drying process on physico-chemical properties of nanoparticles were investigated. The release of insulin from the nanoparticles was studied in vitro. The mechanism of the release of insulin from different types of nanoparticles was determined using curve fitting. The secondary structure of the insulin released from the nanoparticles was analyzed using circular dichroism and the cell cytotoxicity of nanoparticles on a Caco-2 cell line was determined. Ex vivo studies were performed on excised rat jejunum using Frantz diffusion cells. In vivo studies were performed on diabetic male Wistar rats and blood glucose level and insulin serum concentration were determined.
  
  
  RESULTS
  Optimized nanoparticles with proper physico-chemical properties were obtained. The lyophilization process was found to cause a decrease in zeta potential and an increase in PdI as well as and a decrease in entrapment efficiency (EE%) and loading efficiency (LE%) but conservation in size of nanoparticles. Atomic force microscopy (AFM) images showed non-aggregated, stable and spherical to sub-spherical nanoparticles. The in vitro release study revealed higher release rates for lyophilized compared to non-lyophilized nanoparticles. Cytotoxicity studies on Caco-2 cells revealed no significant cytotoxicity for prepared nanoparticles after 3-h post-incubation but did show the concentration-dependent cytotoxicity after 24 h. The percentage of cumulative insulin determined from ex vivo studies was significantly higher in nanoparticles prepared from quaternized aromatic derivatives of chitosan. In vivo data showed significantly higher insulin intestinal absorption in nanoparticles prepared from methylated N-(4-N, N-dimethylaminobenzyl) chitosan nanoparticles compared to trimethyl chitosan.
  
  
  CONCLUSIONS
  These data obtained demonstrated that as the result of optimized physico-chemical properties, drug release rate, cytotoxicity profile, ex vivo permeation enhancement and increased in vivo absorption, nanoparticles prepared from N-aryl derivatives of chitosan can be considered as valuable method for the oral delivery of insulin.
authors.affiliation: Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences , Tehran , Iran .
authors.forename: Reza
authors.initials: R
authors.lastname: Mahjub
authors.forename: Moojan
authors.initials: M
authors.lastname: Radmehr
authors.forename: Farid Abedin
authors.initials: FA
authors.lastname: Dorkoosh
authors.forename: Seyed Naser
authors.initials: SN
authors.lastname: Ostad
authors.forename: Morteza
authors.initials: M
authors.lastname: Rafiee-Tehrani
mesh_terms: D000284:Administration, Oral; D000818:Animals; D001786:Blood Glucose / Q000187:drug effects / Q000378:metabolism; D018938:Caco-2 Cells; D048271:Chitosan / Q000008:administration & dosage* / Q000737:chemistry; D004337:Drug Carriers / Q000008:administration & dosage* / Q000737:chemistry; D016503:Drug Delivery Systems / Q000379:methods*; D005612:Freeze Drying; D006801:Humans; D007328:Insulin / Q000008:administration & dosage* / Q000737:chemistry; D007583:Jejunum / Q000187:drug effects / Q000378:metabolism; D008297:Male; D053758:Nanoparticles / Q000008:administration & dosage* / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar
chemical_list: D001786:Blood Glucose; D004337:Drug Carriers; D007328:Insulin; D048271:Chitosan
keywords: Caco-2 cell cytotoxicity; N-dimethylaminobenzyl) chitosan; circular dichroism; ex vivo and in vivo studies; insulin oral delivery; methylated N-(4-N; methylated N-(4-pyridinyl) chitosan; methylated N-(benzyl) chitosan